The present invention relates to a reagent for detecting and monitoring viral infections, such as those caused by the Epstein-Barr virus or EBV, which is in particular the causative agent of infectious mononucleosis, by the hepatitis C virus (HCV) or by the human immunodeficiency virus (HIV) and to its applications for the detection of a viral infection, in particular of an EBV infection, at any stage of the infection (primary infection, healthy carriers and induced tumours).
EBV is a herpesvirus which preferably infects the B lymphocytes and the epithelial cells.
This lymphocryptic virus escapes immunological surveillance. In general, the virus is carried by healthy carriers, with no special symptoms; however, under immunodeficient conditions, such as AIDS or chemotherapy following a transplant, as well as in endemic regions, such as in Africa or in Asia, the oncogenic potential of EBV is freed and leads to the emergence of various tumours, such as African Burkitt""s lymphoma, undifferentiated carcinoma of the nasopharynx, certain lymphomas and Hodgkin""s disease.
At present the serodiagnosis of EBV is carried out either by the Paul-Bunnell reaction (detection of heterophilic antibodies), a simple and rapid but nonspecific method (LINDERHOLM M. et al., J. Clin. Microbiol., 1994, 32, 1, 259-261), by immunofluorescence (IF) tests which are specific for each class of antigens (VCA, EA and EBNA).
The development of a simpler and less expensive diagnostic test using an immunoenzymatic method such as the ELISA method is in this context particularly desirable and several tests have thus been proposed (M. GORGIEVSKY-HRISOHO et al., J. Clin. Microbiol., 1990, 28, 2305-2311; J M. MIDDELDORP et al., J. Virol. Methods, 1988, 21, 133-159).
The immunoenzymatic methods proposed in the prior art exhibit, for the majority, the major disadvantage of lacking detection sensitivity (M. GORGIEVSKY-HRISOHO et al., cited above; J M. MIDDELDORP et al., cited above). To solve this problem of lack of sensitivity, some authors have proposed using various antigens, in combination; such combinations increase the sensitivity of the test in which these combinations are used, but not the specificity. The combinations of antigens which have been proposed in the prior art comprise, for example, the antigen p47-52 (major antigen EA-D, encoded by the open reading frame called BMRF1, SWISS-PROT accession No. P03191), the antigen p38 (encoded by the open reading frame called BALF2, SWISS-PROT accession No. P03227) and the antigen gp125 or gp110 (encoded by the open reading frame called BALF4, SWISS-PROT accession No. P03188) (W. M. J. Van GRUNSVEN et al., J. Virol., 1993, 67, 3908-3916).
More recently, it has been shown that an 18 kDa capsid antigen (VCAp18), encoded by the open reading frame BFRF3 (SWISS-PROT accession No. P14348), is recognized by healthy carriers of EBV in immunoblot analysis and appears not to exhibit homologous sequences with the other human herpesviruses, unlike the major proteins, such as VCAp40 (sequence encoded by the open reading frame called BdRF1, SWISS-PROT accession No. P03219) and gp125/110 (R. BAER et al., Nature, 1984, 310, 207-211; M. S. CHEE et al., Curr. Topics Microbiol. Immunol., 1990, 154, 125-170; A. J. DAVIDSON, J. Gen. Virol., 1986, 67, 1759-1816). The map of the antigenic domains of the antigen VCAp18 has been established and the major antigenic domain has been localized in the C-terminal region of the protein (W. M. J. Van GRUNSVEN et al., J. Infect. Dis., 1994, 170, 13-18).
However, ELISA tests carried out with this capsid antigen VCAp18 have the disadvantage:
of not exhibiting sufficient sensitivity and specificity to detect all the anti-VCA Ig""s produced (false-positives and/or false-negatives); in particular, as regards the false-negatives, they are essentially due to the small size of the synthetic peptide VCAp18/SEQ ID No. 2 (24 amino acids, SEQ ID No. 2 of the sequence listing), which may consequently not be recognized by all the VCA-positive individuals and/or because of the heterogeneity of the reactivity of the antibodies towards the synthetic peptide, compared with the same peptide, in its natural environment,
of not allowing differential diagnosis of the various stages of the EBV infection and/or of the pathologies induced by EBV, depending on the isotype profile of the Ig""s produced (IgG, IgA and IgM), despite the use of the C-terminal fragment of the VCAp18 protein (SEQ ID No. 1 of the sequence listing), as reagent.
A similar situation is encountered with other viruses, such as HCV or HIV; indeed, in general, the use of one or more immunodominant fragments does not exhibit sufficient sensitivity to avoid false-negatives.
Accordingly, the Applicant company set itself the objective of providing a new reagent for the detection of viral infections, capable of being used in immunoenzymatic tests, which is both specific and sensitive and which makes it possible to obtain a gain in sensitivity of at least 15 to 30% compared with the prior art reagents.
Accordingly, the Applicant company also set itself the objective of providing a new reagent for the detection of EBV infections, capable of being used in immunoenzymatic tests, which is both specific and sensitive and which allows differential diagnosis of the stage of the infection and/or of the pathology, depending on the prevalent isotype; indeed, the presence of human anti-VCA IgMs is essentially the sign of a primary infection, the presence of human anti-VCA IgGs is essentially the sign of a past infection (healthy carriers, generally), whereas the presence of human anti-VCA IgAs suggests the emergence of a tumour. Such a reagent is more suitable for the requirements of practical use than the prior art reagents in the context of immunoenzymatic tests, in particular of the ELISA type.
The subject of the present invention is a reagent for the diagnosis of an infection caused by a virus, characterized in that it comprises essentially a mixture consisting of (1) an immunodominant fragment of a protein of the said virus comprising at most 60 amino acids, preferably between 20 and 30 amino acids and (2) a mixture (called mixotope) of convergent combinatory peptides derived from the said immunodominant fragment, which peptides are obtained by complete or partial artificial degeneration of the said immunodominant fragment by systematic or partial replacement of each amino acid with another according to a suitable replaceability matrix.
For the purposes of the invention, mixotope is understood to mean the mixture of all the combinatory peptides obtained from the selected immunodominant fragment by artificial or constructed degeneration; they are preferably obtained during a single synthesis and represent the peptide antigen and its variability in its antibody population recognition function; various mixotopes may be obtained from the same peptide; the factors which are involved in the constitution of a mixotope are:
on the one hand, the percentage degeneration of the native immunodominant fragment selected (total or partial degeneration); the conserved amino acids (isolated or forming a sequence), in the case of a partial degeneration, are preferably, as regards EBV, those which are involved in the structuring of the VCAp18 protein and
on the other hand, the mode of selection of the substitution of the amino acids of the said native immunodominant fragment; for each position of the sequence of the native immunodominant fragment chosen, the amino acid substitution is selected on the basis of the replacement matrix established by H. M. GEYSEN et al., (J. Mol. Recog., 1988, 1, 32-41), or modified, as illustrated in FIG. 16, taking into account the tolerance of the antibody recognition, depending on the amino acid substitution in the linear epitopes: for a given position, the amino acids exhibiting the highest percentage of xe2x80x9creplaceabilityxe2x80x9d are preferably chosen. However, it is preferable to take into account the conformation of the natural epitopes, before degeneration.
The mixotope for the purposes of the present invention, consisting of convergent combinatory peptides derived from a native immunodominant fragment, therefore represents an artificial and non-natural degeneration of the native structure by the systematic or partial replacement of each amino acid with another derived from the GEYSEN replaceability matrix or from the matrix according to FIG. 16.
Also for the purposes of the invention, constructed replaceability matrix is understood to mean a matrix which does not reproduce the natural variations or the variations frequently observed in evolution; the GEYSEN replaceability matrix or the matrix according to FIG. 16 are particularly suitable.
Surprisingly, the reagents according to the invention make it possible to obtain reliable, reproducible, very sensitive and very specific results since the combinations according to the invention exhibit a synergistic effect in the detection of the antibodies induced by viruses; in particular, a gain in sensitivity of 15 to 30% is generally obtained regardless of the virus (HIV, HCV, EBV, for example).
The subject of the present invention is also a diagnostic reagent according to the invention, characterized in that, for the diagnosis of EBV infections, it comprises essentially a mixture consisting of (1) a C-terminal fragment of the 18 kDa viral capsid antigen (protein VCAp18, SEQ ID No. 1) of the Epstein-Barr virus (EBV) comprising at most 60 amino acids, preferably between 20 and 30 amino acids and (2) a mixture (called mixotope) of convergent combinatory peptides derived from the said C-terminal fragment or peptide VCAp18.
According to an advantageous embodiment of the reagent according to the invention, the said C-terminal fragment of the VCAp18 protein is selected from the group consisting of the peptides VCAp18/SEQ ID No. 2, VCAp18/SEQ ID No. 3, VCAp18/SEQ ID No. 4 and VCAp18/SEQ ID No. 5.
Unexpectedly, the combination of a C-terminal fragment of the VCAp18 protein with a mixotope, derived from the said fragment, and regardless of the configuration of the C-terminal fragment chosen, significantly enhances the sensitivity and specificity of the immunoenzymatic serodiagnosis of EBV; indeed, if an ELISA test using a reagent according to the invention as defined above is compared with an ELISA test using only a peptide VCAp18, a 100% sensitivity and a 100% specificity are effectively obtained with the reagent according to the invention because of the binding of all the anti-VCA Ig""s (G, A and M) to the said reagent.
Surprisingly, the reagents according to the invention make it possible to obtain reliable, reproducible, very sensitive and very specific results because the combinations according to the invention exhibit a synergistic effect in the detection of all the human anti-VCA Ig""s produced.
The mixture of degenerate peptides which is artificially produced during the same synthesis is combined with the native peptide. Such a combination makes it possible, unexpectedly, to increase the reactivity of the mixture of antigens which is produced with respect to the antibodies naturally induced by the virus.
Under these conditions, it is possible to obtain sensitivities of the order of 100% and 97%, respectively, in the populations of post-infected and primary-infected subjects, while preserving 100% specificity in all the tests carried out.
For example:
for the peptide VCAp18/SEQ ID No. 2, it is thus possible to obtain the following three mixotopes:
the mixotope corresponding to a degeneration of the entire sequence of the peptide VCAp18/SEQ ID No. 2, called hereinafter mixotope MIXO,
the mixotope in which the proline residues of the peptide VCAp18/SEQ ID No. 2 are preferentially conserved, the latter being involved in particular in the conformation; the said mixotope, called hereinafter mixotope MIXO(P), corresponds to a partial degeneration of the sequence of the peptide VCAp18/SEQ ID No. 2 and
the mixotope in which both the proline residues and the sequence Gly Ser Gly Gly Gly Gly (SEQ ID No. 6) of the peptide VCAp18/SEQ ID No. 2 are preferentially conserved; the mixotope, called hereinafter mixotope MIXO(P,G), corresponds to a partial degeneration of the sequence of the peptide VCAp18/SEQ ID No. 2,
for the peptide VCAp18/SEQ ID No. 3, it is thus possible to obtain the following mixotopes:
the mixotope corresponding to a degeneration of the entire sequence of the peptide VCAp18/SEQ ID No. 3,
the mixotope in which the proline residue and/or the serine residues of the repetitive sequence of the peptide VCAp18/SEQ ID No. 3 are preferentially conserved; the said mixotope corresponds to a partial degeneration of the sequence of the peptide VCAp18/SEQ ID No. 3,
the mixotope in which the proline residue and the alanine residues of the repetitive sequence of the peptide VCAp18/SEQ ID No. 3 are preferentially conserved; the said mixotope corresponds to a partial degeneration of the sequence of the peptide VCAp18/SEQ ID No. 3 and
the mixotope in which both the proline residue, the serine residues and the alanine residues of the peptide VCAp18/SEQ ID No. 3 are preferentially conserved,
for the peptide VCAp18/SEQ ID No. 4, it is thus possible to obtain the following mixotopes:
the mixotope corresponding to a degeneration of the entire sequence of the peptide VCAp18/SEQ ID No. 4,
the mixotope in which the serine, proline and/or alanine residues and/or the sequence Gly Ser Gly Gly Gly Gly (SEQ ID No. 6) and/or the sequence Ala Ala Ala Ser Ala Ala Ala Ala (SEQ ID No. 7) of the peptide VCAp18/SEQ ID No. 4 are preferentially conserved; the said mixotope corresponds to a partial degeneration of the sequence of the peptide VCAp18/SEQ ID No. 4,
for the peptide VCAp18/SEQ ID No. 5, it is thus possible to obtain the following mixotopes:
the mixotope corresponding to a degeneration of the entire sequence of the peptide VCAp18/SEQ ID No. 5,
the mixotope in which the serine and/or alanine residues of the peptide VCAp18/SEQ ID No. 5 are preferentially conserved; the said mixotope corresponds to a partial degeneration of the sequence of the peptide VCAp18/SEQ ID No. 5,
the mixotope in which both the serine residues and the sequence Ala Ala Ala Ser Ala Ala Ala Ala (SEQ ID No. 7) of the peptide VCAp18/SEQ ID No. 5 are preferentially conserved; the said mixotope corresponds to a partial degeneration of the sequence of the peptide VCAp18/SEQ ID No. 5.
The different mixotopes corresponding to a partial degeneration of the chosen peptide VCAp18 conserve a native sequence which is preferably involved in the structuring of the VCAp18 protein.
According to another advantageous embodiment of the reagent according to the invention, it is attached to a solid support, preferably microtitre plates.
According to another advantageous embodiment of the reagent according to the invention, the C-terminal peptide:mixotope ratio in the mixture is between 1:10 and 1:100.
The subject of the present invention is also a method for the diagnosis of a viral infection, by an immunoenzymatic method, characterized in that it uses a diagnostic reagent according to the invention.
In particular, as regards EBV infections, the subject of the present invention is also a method of diagnosis by an immunoenzymatic method, characterized in that it comprises:
bringing a serum to be analysed into contact with a reagent as defined above,
the addition of anti-human Ig antibodies coupled to an enzyme, and
the qualitative and/or quantitative revealing of the anti-VCA antibodies which may be present in the serum to be analysed by addition of the enzyme substrate.
According to an advantageous embodiment of the said method, it comprises:
the attachment of a reagent according to the invention onto a support, such as a microtitre plate,
the addition of the serum to be analysed,
the detection of the attachment of the anti-VCA antibodies present in the said serum by addition of anti-human Ig (G-A-M) antibodies coupled to an enzyme, and
the qualitative and/or quantitative revealing in a spectrophotometer by addition of the enzyme substrate.
The subject of the present invention is also a method for the surveillance and differential detection of the stages of an EBV infection by an immunoenzymatic method, characterized in that it comprises:
the attachment of a reagent according to the invention onto a support, such as a microtitre plate,
the addition of the serum to be analysed,
the detection of the attachment of the anti-VCA antibodies, which may be present in the said serum, by the addition of anti-human Ig antibodies coupled to an enzyme, which antibodies are selected from the group consisting of the anti-human IgG antibodies, the anti-human IgM antibodies and the anti-human IgA antibodies, and
the qualitative or quantitative revealing in a spectrophotometer by the addition of the enzyme substrate.
The primary infection induces the formation of antibodies directed against the different antigens of EBV; they are in particular the antibodies directed against the viral capsid antigen (VCA), the antibodies directed against the nuclear antigen (EBNA), the antibodies directed against the early antigen (EA) and the antibodies directed against the membrane antigen (MA).
These different antibodies do not appear at the same stage of the infection; in particular, the anti-VCA Ig""s are produced very early and remain present during the entire life of the host. This means that the detection of the anti-VCA IgMs and IgGs in the human serum reinforces the diagnostic value (particularly advantageous), in order to establish a primary infection with EBV.
Depending on the isotype of the anti-VCA Ig""s which is detected in the serum to be analysed, it is possible to distinguish the healthy carriers of the virus (prevalence of the IgGs), the primary infections (prevalence of the IgMs) or the emergence of a tumour, in particular carcinoma of the nasopharynx (prevalence of the IgAs).
Surprisingly, the reagent according to the invention effectively makes it possible to detect the anti-VCA IgAs.
The subject of the present invention is also a method for the early detection of the cancer of the nasopharynx by an immunoenzymatic method, characterized in that it comprises:
the attachment of a reagent according to the invention onto a support, such as a microtitre plate,
the addition of the serum to be analysed,
the detection of the attachment of the anti-VCA antibodies, which may be present in the said serum, by the addition of anti-human IgA antibodies coupled to an enzyme, and
the qualitative or quantitative revealing in a spectrophotometer by the addition of the enzyme substrate.
The subject of the present invention is, in addition, a kit or box for the diagnosis of a viral infection, characterized in that it comprises at least one diagnostic reagent according to the invention.